Project Summary/Abstract Human cytomegalovirus (HCMV) infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can result in severe and debilitating consequences. The majority of the US Food and Drug Administration approved anti-HCMV drugs target the viral polymerase and resistance to those drugs has appeared. Therefore, anti-HCMV drugs from novel targets are needed for use in combination with, or instead of, current polymerase inhibitors. The long-term goal of this proposal is to develop structurally novel direct-acting antivirals against HCMV targeting aspects of virus replication outside of those for currently approved drugs. Towards this end, a viral ATPase/endonuclease (pUL89) critically involved in viral genome packaging and virus assembly has been chosen as a target for antiviral intervention. The objective of this investigation is to identify, optimize and characterize pUL89 inhibitor scaffolds. To accomplish these goals, the following specific aims will be pursued: 1. Develop and implement screening assays to identify pUL89 and HCMV inhibitors. These assays include biochemical assays for pUL89 function as well as HCMV replication assays in cell culture. 2. HCMV pUL89 inhibitor hit generation and optimization. Hit generation will center around the focused screening of in-house synthetic small molecules and select compound libraries. Confirmed hits will be optimized via analogue synthesis, iterative SAR and in vitro ADME assays to generate potent inhibitors with favorable physicochemical properties. 3. Characterization of advanced inhibitors using virological, structural and pharmacokinetic techniques. Optimized inhibitor binding to pUL89 will be analyzed and refined using structural studies. Detailed virological experiments will be performed to characterize the mechanism of action, verify the target of these inhibitors and document their ability to work with current FDA approved drugs and against resistant virus. In addition, in vivo pharmacokinetic parameters of the optimized inhibitors will be determined and used to improve drug-like properties. At the conclusion of the studies outlined in this proposal, a better understanding of the mechanism of action and role of pUL89 endonuclease activity in virus replication will be gained and multiple potent preclinical drug candidates targeting pUL89 will be developed.